L-deprenyl for treating immune system dysfunction and compositions for same

ABSTRACT

Use of L-deprenyl for treatment of immune system dysfunction for mammals.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 07/975,284 filed on Nov.12, 1992, now U.S. Pat. No. 5,276,057, which is a CIP of Ser. No.07/643,452 filed on Jan. 18, 1991, now abandoned, which is a CIP of Ser.No. 07/576,011, filed on Aug. 31, 1990, now U.S. Pat. No. 5,151,449.

BACKGROUND THE INVENTION

L-deprenyl is a selective monoamine oxidase B (MAO-B) inhibitor, whichis widely used as an adjunct in the treatment of Parkinson's disease.While its most common usage is for the treatment of Parkinson's disease,L-deprenyl was originally developed as an antidepressant agent. Recenttesting has indicated that L-deprenyl may have some effect on increasingsexual response in aging animals, and also may have some effect, atleast in rats in increasing life expectancy. However, to date L-deprenylhas only been medically approved by regulatory agencies for use as atreatment for Parkinson's disease in humans.

Parkinson's disease is a distinct disease associated with depletion ofdopamine in the basal ganglia area of the brain, and the cardinal signsrelate to motor dysfunction. Parkinson's disease is one of the mostcommon causes of disability in older humans, with approximately 1% ofpersons being afflicted after age 60 (Jankovic, J. Parkinsonism. In:Conn's Current Therapy 1992; R. Rakel (ed.), W. B. Saunders Co., page880, 1992). The cardinal signs involve various motor deficits, sometimescomplicated by either neurobehavioral or other non-motor problems.Typical pathologic lesions of Parkinson's disease include neuronal"dropout" and the presence of Lewey bodies in the substantia nigra. Thedisease is considered progressive, despite advances in therapy. L-dopareplacement therapy is the primary medical treatment, althoughl-deprenyl and anticholinergics are also used in Parkinson's diseasetherapy.

Parkinson's disease is normally not thought of as a disease of immunesystem dysfunction although there are some literature reports discussingimmune system abnormalities in Parkinson's disease. However, manyParkinson's Disease patients do show quite normal immune systemfunction. Furthermore, there are no known reports of using l-deprenyl totreat immune system abnormalities. The limited reports of immune systemdysfunction in Parkinsonians have been inconsistent, and tend toidentify immune system problems in chronically PD-afflicted persons, thesame persons who have received extended therapy with 1-dopa. It is knownthat 1-dopa may cause T-lymphocyte functional abnormalities in mice(Boukhris, W., Kouassi, E. et al. Impaired T-Dependent Immune Responsein L-Dopa Treated BALB/C Mice. J. Clin. Lab. Immunol. 23(4). p. 185-189,1987). Thus, the immune system abnormalities seen in some cases of PDmay be caused by chronic 1-dopa therapy. Since l-deprenyl is used toaugment 1-dopa therapy, it would be reasonable to conclude that1deprenyl therapy would increase rather than decrease any immune systemabnormalities in PD. To our knowledge, there are no published reportsdiscussing l-deprenyl and its effect on the immune system function ineither health or disease states, and there are no published reportsdescribing the treatment of immune system dysfunction in any specieswith l-deprenyl.

Immune system dysfunction can and does occur at any stage of life due toa variety of causes, including aging (see p. 10-13). In many animals,i.e., man, dogs, monkeys, etc., immune system dysfunction, particularlyas the animal ages, can substantially increase disease risk, which canthreaten life itself. It is therefore important for over all well beingto effectively treat any immune system dysfunction. Doing so will retardthe risk of diseases known to affect animals with lowered immuneresponse.

There is, therefore, a continuing and real need for the development ofmedications which treat immune system dysfunction.

In the grandparent application of co-inventor Milgram, now U.S. Pat. No.5,151,449, there are disclosed certain uses of L-deprenyl forretardation of normal age dependent deterioration of renal function,retardation of normal degeneration of cognitive abilities, and forretardation of age dependent weight loss. In accordance with theimprovement invention of parent application Ser. No. 07/643,452, it hadbeen discovered that L-deprenyl will also assist in maintenance ofthyroid function, adrenal function, immune system function andmaintenance of body composition in aging mammals, providing that certaindoses and levels of use were maintained. It may also be useful intreating Cushing's disease, (see Ruehl, Therapeutic Effect of L-DeprenylIn The Management of Pituitary-Dependant Hyperadrenocorticism (Cushing'sDisease) filed Mar. 27, 1992).

While L-deprenyl is a known compound, it has never before beendemonstrated effective and used at any level to treat immune systemdysfunction.

Like most drugs, L-deprenyl can have diverse physiological effects whichare completely dependent upon the dose administered. In accordance withthe present invention, L-deprenyl can be used for successful methods oftreatment for immune system dysfunction, providing that it is used atthe dosage levels mentioned herein, and providing it is administered atthe periodic intervals and for the length of time mentioned herein.Obviously, when different dosages and levels of treatment are used, theresults expressed herein may not be achieved. In fact, at higher doses,adverse behavioral effects may be encountered.

Accordingly, a primary objective of the present invention is to developa dosage regimen for use of L-deprenyl to treat immune systemdysfunction in mammals.

The method and means of accomplishing this as well as other primaryobjectives of the present invention will be apparent from the detaileddescription which will follow hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the IgG and IgA levels after the treatment of Example3.

SUMMARY OF THE INVENTION

The present invention relates to the process of using a known compound,L-deprenyl, for treating immune system dysfunction in mammals. Inparticular, at the dosage levels described herein, providing that thedosage is used for at least the periods of time expressed herein, thereis an observed and measurable effect upon a dysfunctioning immunesystem. The treatment is shown to be useful for domesticated pets suchas dogs, as they increase in age, but would be expected to have a likeutility in any mammalian species, including humans.

DETAILED DESCRIPTION OF THE INVENTION

As earlier stated, the compound that is useful for the method orprotocol of the present invention is a known compound, L-deprenyl.L-deprenyl has the formula(-)-N-a-dimethyl-N-2-propynylbenzene-ethanamine. It can be illustratedby the following graphic formula: ##STR1##

L-deprenyl also is at times referred to as (-)deprenyl to illustratethat it is a levorotary isomer. Typically, it is provided in apharmaceutically acceptable salt form thereof such as the hydrochloridesalt.

As used here, pharmaceutically acceptable salt form thereof, means thefollowing. Acceptable for use in the pharmaceutical or veterinary art,being nontoxic or otherwise not pharmaceutically or veterinaryunacceptable. "Acceptable salt form thereof" means salts formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like, and as well organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, etc.

Administration of the therapeutically active compound l-deprenyl toachieve physiological results of the present invention can be via any ofthe accepted modes of administration for systemically active substances.These methods include oral, parenteral, and otherwise systemic, aerosol,and topical forms, as well as sustained release systems, etc.

The compositions of the present invention may be any of those known inthe pharmaceutical and veterinary arts which are suitable for the methodof administration and dosage required in any particular circumstance. Inthe case of both pharmaceutical and veterinary applications, suchcompositions may include tablets, pills, capsules, powders, aerosols,suppositories, skin patches, parenterals, and oral liquids including oilaqueous suspensions, solutions and emulsions. It may include long actinginjectables and sustained release devices.

When the dosage is in solid form, solid pharmaceutical carriers such asstarch, sugar, talc, mannitol, povidone, magnesium stearate, and thelike may be used to form powders. Lactose and mannose are the preferredsolid carrier. The powders may be used as such for direct administrationto a patient or, instead, the powders may be added to suitable foods andliquids, including water, to facilitate administration.

The powders also may be used to make tablets, or to fill gelatincapsules. Suitable lubricants like magnesium stearate, binders such asgelatin, and disintegrating agents like sodium carbonate in combinationwith citric acid may be used to form the tablets.

Unit dosage forms such as tablets and capsules may contain any suitablepredetermined amount of l-deprenyl, advisably as a nontoxic acidaddition salt, and may be administered one or more at a time at regularintervals as later described. Such unit dosage forms, however, shouldwith a broad range guideline contain a concentration of 0.1 mg/kg to 5.0mg/kg of the active l-deprenyl.

A typical tablet for the specified uses mentioned herein in a 25 kg dogmay have the composition:

    ______________________________________                                                       Mg.                                                            ______________________________________                                               1. L-deprenyl                                                                            25                                                                 2. Mannitol                                                                             100                                                                 3. Stearic acid                                                                          3                                                           ______________________________________                                    

A granulation is made from the mannitol. The other ingredients are addedto the dry granulation and then the tablets are punched.

Another tablet may have the composition:

    ______________________________________                                                      Mg.                                                             ______________________________________                                        1. L-deprenyl   25                                                            2. Starch U.S.P.                                                                              57                                                            3. Lactose U.S.P.                                                                             73                                                            4. Talc U.S.P.   9                                                            5. Stearic acid  6                                                            ______________________________________                                    

Powders 1,2 and 3 are slugged, then granulated, mixed with 4 and 5, andtableted.

Capsules may be prepared by filling No. 3 hard gelatin capsules with thefollowing ingredients, thoroughly mixed:

    ______________________________________                                                      Mg.                                                             ______________________________________                                        1. L-deprenyl   25                                                            2. Lactose U.S.P.                                                                             200                                                           3. Starch U.S.P.                                                                              16                                                            4. Talc U.S.P.   8                                                            ______________________________________                                    

As earlier expressed, physiological functions affected by the treatmentherein with L-deprenyl are necessarily dosage dependent. Put anotherway, like most drugs, l-deprenyl has diverse physiological effectsdepending upon the dose administered. Unless the dose administered iswithin the levels set forth herein, the desired effects on immunefunction, without adverse effects, are not achieved.

Immune reactions involve the coordinated efforts of several types ofcells, such as T and B lymphocytes and macrophages. Substantial evidenceexists in several species that immune dysfunction occurs with increasingage and that impaired T-cell function may be one of the mechanisms forthe decline. While some changes in B lymphocyte function have beenreported, it is difficult to dissociate these effects from concomitantchanges in T-Cells. Also, little or no evidence supports changes inantigen presenting cells (macrophages) with increasing age. On the otherhand, a number of investigators have shown a substantial decline in mostmeasures of T-cell function with age.

The most studied of the immune parameters is lymphocyte proliferation.The proliferative capacity of both T- and B-cells has been found todecline substantially with increasing age (Morgan et al., "The immuneresponse in aged C57BL/6 mice. I. Assessment of lesions in the B-celland T-cell compartments of aged mice utilizing the Fc fragment-mediatedpolyclonal antibody response", Cellular Immunology 63, 16-27, 1981;Abraham et al., "Reduced in vitro response to concanavalin A andlipopolysaccharide in senescent mice: A function of reduced number ofresponding cells", European Journal of immunology, 7, 301-304, 1977;Hefton et al., "Immunologic studies of aging. V. Impaired proliferationof PHA responsive human lymphocytes in culture", Journal of Immunology125, 1007-1010, 1980). These studies utilize splenocytes which aremitogenically challenged with phytohemaglutin (PHA) or concanavalin-A invitro in the presence of 3H-thymidine or bromodeoxyuridine (BrdU).Thymidine or BrdU uptake by the cells is then measured and reflects thenumber of cells entering mitosis. Also, upon activation by mitogens orantigens, T-cells secrete a number of antigen-non-specific growth andmaturation factors, collectively termed lymphokines. One of these,Interleukin-2 (IL-2), seems to be required for T-cell division and playsa role in B-cell growth as well. T-cells from old mice and humans havebeen found to secrete diminished amounts of IL-2 when triggered bymitogens, alloantigens or foreign antigens (Gillis et al.,"Immunological studies of aging. Decreased production of and response toT cell growth factor by lymphocytes from aged humans", Journal ofClinical Investigation 67, 937-942, 1981; Miller and Stutman, "Decline,in aging mice, of the anti-TNP cytotoxic T cell response attributable toloss of Lyt-2, IL-2 producing helper cell function", European Journal ofImmunology 11, 751-756, 1981; Nagel et al., "Decreased proliferation,interleukin 2 synthesis, and interleukin 2 receptor expression areaccompanied by decreased mRNA expression inphytohemagglutinin-stimulated cells from elderly donors", Journal ofClinical Investigation 81, 1096-1102, 1988; Thomas and Weigle, "Partialrestoration of Con A-induced proliferation, IL-2 receptor expression,and IL-2 synthesis in aged murine lymphocytes by phorbol myristateacetate and ionomycin", Cellular Immunology 114, 1-11, 1988). Further,several studies have shown that aging leads not only to poor productionof IL-2, but also to diminished responsiveness to this growth factor.Thus, addition of exogenous IL-2 leads to only a partial restoration offunction in immune cells from old animals (Gillis et al., "Immunologicalstudies of aging. Decreased production of and response to T cell growthfactor by lymphocytes from aged humans", Journal of ClinicalInvestigation 67, 937-942, 1981; Gilman et al., "T lymphocytes of youngand aged rats. II. Functional defects and the role of interleukin-2",Journal of Immunology 128, 644-650, 1982; Gottosman et al.,"Proliferative and cytotoxic immune functions in aging mice. III.Exogenous interleukin-2 rich supernatant only partially restoresalloreactivity in vitro", Mechanisms of Ageing and Development 31,103-113, 1985). Also, the number of T-cells able to express IL-2receptors upon stimulation by a mitogenic agent declines with age inboth humans and mice (Negoro et al., "Mechanisms of age-related declinein antigen specific T cell proliferative response: IL-2 receptorexpression and recombinant IL-2 induced proliferative response ofpurified TAC-positive T cells", Mechanisms of Ageing and Development 36,223-241, 1986; Vie and Miller, "Decline, with age, in the proportion ofmouse T cells that express IL-2 receptors after mitogen stimulation",Mechanisms of Ageing and Development 33, 313-322, 1986). Aging,therefore, leads to a decline in both production of and response toIL-2.

It has also been reported that older dogs produce less antibody to anexogenous T-lymphocyte dependent antigen, sheep red blood cells, thanyounger dogs (Bice, D. and Muggenburg, B. "Effect of Age on AntibodyResponses after Lung Immunization", American Revue of RespiratoryDisease. 132:661-665, 1985). This was indicative of the normalage-related decline in immune system function, or age-related immunesystem dysfunction, that is documented in various species, includingdogs, rodents, non-human primates and humans.

The term "mammal" as used herein includes without limitation humans anddomesticated animals such as cattle, horses, swine, sheep, dogs, cats,goats and the like. The tests hereinafter shown in the examples areparticularly illustrative for dogs, but indicate usage for humans aswell as for domesticated animals including cats and other mammals. Thetreatment may even work for birds or fish.

Needless to say, the natural enjoyment of pets would be significantlyincreased in their older age if their general well being and and healthis maintained. This requires a healthy, functioning immune system.

In accordance with the present invention, it is illustrated that immunesystem response can be improved if the animal is treated periodicallywith small but therapeutically effective doses of l-deprenyl.

As hereinafter explained, the dosage regimen to achieve these desirableresults differs considerably from the dosage regimen used in treatingParkinson's disease and may differ in quantity and frequency from thedose levels in the parent application, Milgram, Ser. No. 07/576,011filed Aug. 31, 1990. At the preferred dose recommended for usesdisclosed herein, the levels are up to 25X the amount used for treatingParkinson's disease. In particular, the dosage regimen of the presentinvention shows usage at levels from about 0.1 mg/kg of body weight upto about 5.0 mg/kg of body weight from 1 to 7 times weekly, preferablydaily. Most preferably the dosage level is 0.5-2.0 mg/kg of body weightgiven once daily. Of course it would be known to those in the art thatsustained release systems can be used to provide less frequentadministration to achieve the required dosage level.

It is not known precisely why the use of l-deprenyl at the dosage levelsand periodicity expressed herein achieves these results. As explained inthe examples below, analysis of blood specimens suggests that thel-deprenyl treatment may have a direct effect on the immune systemspecifically. It is important that the dosage be at levels expressedherein rather than at Parkinson's disease levels, otherwise nobeneficial effects may be achieved.

EXAMPLES

The following examples provide illustrative evidence of the results fortreating mammals with l-deprenyl for immune system dysfunction.

EXAMPLE 1 Example of Maintaining Peripheral Blood White Cell andLymphocyte Counts in Aging Fischer Rats

Sixty two male Fischer 344 rats were obtained at 21-23 months of age.The animals were allowed free access to food and water. L-deprenyl wasinjected subcutaneously every other day at a dose of 0.25 mg/kg bodyweight, starting at 24-25 months of age. Thirty one rats were given thel-deprenyl, and thirty one rats served as sham-injected controls (givensaline).

Blood samples were collected on all rats at the beginning of theexperiment and again from surviving rats after three months ofl-deprenyl therapy. The blood samples were sent to a local commercialclinical pathology laboratory for routine hematological testing. Adouble blind procedure was followed in the collection and analysis ofthe blood samples.

As these F344 rats become older, there is a trend for abnormal increasesin peripheral blood lymphocytes, which may be associated with andindicative of immune system dysfunction. In the rats receivingl-deprenyl, there was a distinct inhibition in the proliferation ofperipheral blood lymphocytes, which might be expected with age-relatedimmune system dysfunction. The data is presented in Table 1. Theseresults indicate that chronic l-deprenyl therapy may be beneficial inage-related immune system dysfunction in rats.

                  TABLE 1                                                         ______________________________________                                        Baseline (24-25 months)                                                                           27-29 Months                                              Control       l-Deprenyl                                                                              Control    L-Deprenyl                                 ______________________________________                                        WBC     5.32 ± .94                                                                           5.20 ± .43                                                                           15.89 ± 5.03                                                                        8.45 ± 1.6                            count*                                                                        Lympho- 2.16 ± .31                                                                           2.15 ± .29                                                                           10.64 ± 4.47                                                                        4.12 ± 1.27                           cyte count*                                                                   ______________________________________                                         *numbers are × 10 to the 6th per ml. of blood                      

EXAMPLE 2

Example of Treating Age-Related Immune System Dysfunction

Old (12 to 16 years) laboratory beagles of either sex were divided intotwelve animals per group. Similar laboratory beagles have been shown tohave age-related immune system dysfunction, as manifested bysignificantly lower antibody responses to the exogenous antigen, sheepred blood cells (Bice, D. and Muggenburg, B. "Effect of Age on AntibodyResponses after Lung Immunization", Am. Rev. Resp. Dis. 132: 661-665,1985). The experimental group received 1 mg/kg of l-deprenyl every dayvia oral tablets for approximately six months.

Lymphocyte subset data was developed to determine whether theage-related immune system dysfunction characterized by a decrease in thehelper/suppressor lymphocyte ratio might be treated by l-deprenyladministration. This data was developed using monoclonal antibodies tospecific lymphocyte cell surface antigens using standard cell sorting(flow cytometry) techniques (Moore, P. F. et al. Monoclonal AntibodiesSpecific for Canine CD4 and CD8 define functional T lymphocyte subsetsand high-density expression of CD4 by canine neutrophils, TissueAntigens 40: 75-85, 1992.)

The below set forth data Table 2 provides the results. Note particularlythe CD4/CD8 ratio of 2.5 in young dogs; 1.37 in old untreated dogs; and1.89 in old l-deprenyl treated dogs. This example demonstrates that theperipheral blood lymphocyte "helper cell" to "suppressor cell" ratio,which often decreases with increasing age (and which may help explainage-related immune system dysfunction), is amenable to chronicl-deprenyl therapy. Treating the age-related decrease in thehelper/suppressor cell ratio with l-deprenyl therapy may partiallyexplain why older dogs with immune system dysfunction are able to mounta statistically increased humoral antibody response to an T-lymphocytedependent antigen such as tetanus toxoid (see. Example 3, below).

                  TABLE 2                                                         ______________________________________                                        Effect of L-Deprenyl on T-Lymphocyte Subsets                                  in Aging Dogs                                                                             T-Lymphocyte Subsets                                                                                 CD4/CD8                                    Group         CD4        CD8       Ratio                                      ______________________________________                                        1. Young Control Dogs                                                                       53.6 ± 6.6                                                                            21.8 ± 4.3                                                                           2.56 ± 0.64                             2. Old Control Dogs                                                                         31.8 ± 6.8                                                                            26.3 ± 9.2                                                                           1.37 ± 0.56                             3. L-Deprenyl Treated                                                                        39.4 ± 11.3                                                                          23.7 ± 9.1                                                                           1.89 ± 0.86                               Old Dogs                                                                    ______________________________________                                    

EXAMPLE 3 Example of Humoral Antibody Response to Tetanus Toxoid

Old (12 to 16 years) laboratory beagles of either sex were divided intotwelve animals per group. Similar laboratory beagles have been shown tohave age-related immune system dysfunction, as manifested bysignificantly lower antibody responses to the exogenous antigen, sheepred blood cells (Bice, D. and Muggenburg, B. "Effect of Age on AntibodyResponses after Lung Immunization", Am. Rev. Resp. Dis. 132: 661-665,1985). The experimental group received 1 mg/kg of l-deprenyl every dayvia oral tablets for approximately six months.

The immune system of each animal was challenged with 0.5 ml ofcommercially available tetanus toxoid by subcutaneous inoculation on day0 and day 21. The inoculations were given on Jul. 27th and Aug. 17th,1992.

The immune response was evaluated using an enzyme-linked immunosorbentassay ("Elisa") using standard methods known in the art (Brice, D. andMuggenburg, B. "Effect of Age on Antibody Responses after LungImmunization", Am. Rev. Resp. Dis. 132: 661-665, 1985).

The immune response evaluation schedule occurred on days 3,5,7,9,11 and14 after the first inoculation and days 2,4,7,9,11 and 14 after thesecond inoculation.

The T-lymphocyte dependent antigen tetanus toxoid was used in thesetests to determine whether or not l-deprenyl administration bolster-edthe systemic immune response in aged dogs. The serum antibody levels(IgA and IgG) were measured. The synthesis and the secretion of theseantibodies in the blood is indicative of the integrity of the immuneresponse and provides a quantitative assessment of the magnitude of theresponse, with higher levels of antibodies representing a more robustsystemic immune system and response and lower antibody levelsrepresenting a relative dysfunction of the immune system.

The data was statistically analyzed using ANOVA (analysis of variance)comparison on individual days and repeated measures ANOVA over theentire time course.

There was no statistically significant difference between theexperimental and the control groups at time zero. After the firstinoculation the l-depreynl treated group exhibited a trend towardgreater levels of specific serum IgG and IgA, although statisticalsignificance was not achieved. However, the experimental dogs that hadbeen given l-deprenyl for six months had statistically significantincreases in specific anti-tetanus serum IgA and serum IgG after thesecond (booster) inoculation as shown in FIGS. 1 and 2.

These results indicate that old dogs that have been given 1 mg/kg ofbody weight of l-deprenyl for approximately six months, have astatistically significant increase in humoral systemic immune response(IgA and IgG) to tetanus toxoid after the booster dose, as compared tocontrol dogs. The administration of l-deprenyl at 1 mg/kg to aged dogsimproved the systemic response to a test foreign antigen, tetanustoxoid. There is therefore a statistically significant, effectivetreatment of immune system dysfunction in these older animals if theyare provided with l-deprenyl at the dose levels of the present inventionfor significant periods of time.

What is claimed is:
 1. A method of treating immune system dysfunction ofhumans by stimulating a secretion selected from the group consisting ofTumor Necrosis Factor alpha, Interleukin-6 and Granulocyte-MacrophageColony Stimulating Factor, said method comprising:administering to thehuman a dose level greater than level used to treat Parkinson's diseaseand an immune response dysfunction treating effective amount of thecompound L-deprenyl, or a pharmaceutically acceptable form thereof, at afrequency level of from about one to about seven times weekly until adesired effect upon deterioration of immune system response function isachieved.
 2. The method of claim 1 wherein the L-deprenyl is thehydrochloride addition salt form thereof.
 3. The method of claim 1wherein the addition level is at a level of from 0.1 mg/kg of bodyweight to 5.0 mg/kg of body weight.
 4. The method of claim 3 wherein theaddition level is from about 0.1 mg/kg of body weight to about 5.0 mg/kgof body weight, dosed from one to seven times weekly.
 5. A method oftreating immune system dysfunction of hormoral antibody response and Tlymphocyte function of mammals, said method comprising:administering tothe mammal a small but humoral antibody response and T lymphocytedysfunction treating effective amount of the compound L-deprenyl, or apharmaceuticall acceptable form thereof, at a frequency level of fromabout one to about seven times weekly until a desired effect upondeterioration of immune system response function is achieved.